【作者】 章述军；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 [目的]戊型肝炎是由戊型肝炎病毒引起的经消化道传播的急性自限性疾病,以往通常发生于卫生条件落后的发展中国家和落后国家,我国是高发地区之一。但是,近年来在一些发达国家如美国、欧洲等地陆续报道了非输入性戊型肝炎的发生,另外,有证据显示戊型肝炎还可以引起人畜共患性疾病,而且在孕妇死亡率可高达20-30%,对社会造成了极大的危害,因此戊型肝炎越来越受到人们的重视。迄今为止,尚无有效的HEV感染的细胞模型和动物模型,因此其感染和发病机制尚不清楚,而且也没有建立统一的诊断标准、分级标准和广泛认可的诊断试剂。本课题旨在通过临床大样本戊型肝炎病例的研究,建立戊型肝炎死亡的预报模型和新的诊断方法,并未后续研究提供临床依据和线索。[方法]1.收集2007-2008年湖北武汉同济医院和传染病院戊型肝炎患者共计210例,所有患者均采集系列血清进行HEV RNA、血清学和生化学指标的检测,随访半年。将存活组与死亡组进行组间比较,对有意义的指标进行单因素和多因素Logistic回归分析,筛选出戊型肝炎患者死亡的危险因素,建立戊型肝炎患者死亡概率的预报模型,并进行评估。2.利用来源于基因Ⅰ型戊型肝炎病毒开放读码框2,并可形成多聚体形式的p239抗原,建立抗HEV-IgA检测的间接酶联免疫吸附诊断试剂盒。利用ROC曲线法确定临界值。然后以2210份临床排除急性戊型肝炎病毒感染血清作为阴性血清盘,245位临床诊断戊型肝炎患者系列血清作为阳性血清盘,评估试剂盒的特异度、灵敏度和临床应用价值。3.构建基因Ⅰ型和Ⅳ型戊型肝炎病毒完整的开放读码框3的真核表达质粒,激光共聚焦显微镜观察ORF3的细胞内定位。MTT法检测其对两个基因型的戊型肝炎病毒ORF3基因对HepG2细胞增殖的影响,对抗放线菌素D损伤细胞的作用,western blotting检测其对HepG2细胞核内NF-κB表达的影响。[结果]1.湖北地区散发性戊型肝炎均为基因Ⅳ型戊型肝炎病毒感染,总病死率为10%(21／210)。男性多发(85.2%),中老年多见：全年均可见到发病,但是以第一季度为高发季节,占到了全年总发病数的50.5%。血清总胆红素(OR值=1.0009,每1μmol/L)、血尿素氮(OR值=1.178,每1mmol/L)和国际标准化比值(OR值=9.216,每1)为戊型肝炎患者死亡的主要危险因素。建立了戊型肝炎患者死亡概率的预报模型,从起病第二周即能较好的预测患者的临床转归,曲线下面积达0.957。2.利用P239抗原成功构建了检测抗HEV-IgA的间接ELISA试剂盒,特异度达到了99.6%(95%置信区间：99.2%-99.8%)。在245例临床诊断戊型肝炎的患者,有84例患者血清中检出HEV RNA。在此84例HEV RNA作为金标准的患者中,抗HEV-IgA、抗HEV-IgM和抗HEV-IgG的阳性率分别为96.3%(81／84)(95%置信区间：89.8%～98.8%)、97.6%(82／84)(95%置信区间：91.7%～99.3%)和88.1%(74／84)(95%置信区间：79.5%～93.4%),并且没有样本抗HEV-IgA和抗HEV-IgM同时阴性的。在245例戊型肝炎患者中,发现9份分别来自于9例患者的血清样本在急性期内存在抗HEV-IgM阴性的时期,但是9份样本抗HEV-IgA均为阳性,其中两例检出HEV RNA。3.成功构建了基因Ⅰ型和Ⅳ型HEV ORF3的真核表达质粒,测序证实构建成功,并能够在转染的HepG2细胞成功表达。激光共聚焦显示HEV ORF3蛋白主要定位与细胞浆内而不能进入细胞核。MTT检测发现基因Ⅰ型和Ⅳ型HEV ORF3基因在正常情况下对HepG2细胞的增殖没有影响,而在放线菌素D处理的HepG2细胞中则能够表现出显著促进存活的作用,且在两个基因型HEV ORF3之间没有显著性差异。Western blotting显示HEV ORF3能够促进HepG2细胞核内P65的表达,且在两个基因型HEV ORF3之间没有显著性差异。【结论】1.武汉地区戊型肝炎仅见基因Ⅳ型HEV感染,HE患者中男性比例高于女性,中老年人为主,第一季度即冬春季为散发性戊型肝炎的高发季节。血清总胆红素、血尿素氮和国际标准化比值是戊型肝炎患者死亡的主要危险因素,初步构建了戊型肝炎患者死亡概率的预报模型,预测价值较好。2.成功构建抗HEV-IgA间接ELISA检测试剂盒,其特异度和在基因Ⅳ型HEV感染患者中的灵敏度均较好,联合抗HEV-IgM检测可使戊型肝炎患者诊断的特异度和灵敏度均达到100%,有助于提高临床上对戊型肝炎诊断的准确度。3.基因Ⅰ型和Ⅳ型HEV ORF3均定位于细胞胞浆中,对于放线菌素D处理的HepG2细胞具有保护作用,并有可能是通过激活NFκB通路而产生作用的。更多还原

【Abstract】 【Objective】Hepatitis E is a disease caused by hepatitis E virus and is transmitted through the digestive tract. Hepatitis E usually occurs in developing countries where health conditions are relatively poor, but an epidemiological survey showed that Hepatitis E can also occur in developed countries among people without a history of travel to areas of high incidence. In recent years, the number of Hepatitis E patients has shown an upward trend. Many developed countries have reported native cases of Hepatitis E. Its mortality rate is much higher than that of Hepatitis A and Hepatitis B, especially in pregnant women whose mortality rate reaches 20-30%. So far, there is no animal and cell model for hepatitis E virus and its pathogenesis is not clear. In addition, diagnostic criteria and reagent is lacking. Therefore, we hope that we can get some clues through the analysis of a large number of patients with hepatitis E.【Methods】1. This study included 210 hepatitis E patients from Department of Infectious Diseases of Wuhan Tongji Hospital within the period of January 2007 to December 2008. The serum samples from all of the patients with hepatitis E was collected for HEV RNA detection, antibody against hepatitis E virus and biochemical indicator. Use the comparison between deceased group and survival group, univariate and multivariate logistic regression to screen the risk factors for death of hepatitis E. Use the risk factors to construct the probability of death equation for hepatitis E. 2. Use the p239 antigen corresponding to aa368-aa606 of the open reading frame 2 of a genotype 1 strain of hepatitis E virus to construct the indirect enzyme linked immunosorbent assay to detect anti HEV IgA. The cut off value is defined by receiver operating characteristic curve analysis. Then, use the 2210 serum samples with elimination of hepatitis E virus infection as the negative control panel and serum samples collected from 245 hepatitis E patients as positive control panel. The specificity, sensitivity and the value of clinical application was evaluated.3. To construct eukaryotic expression vectors for expressing genotypeⅠhepatitis E virus and genotypeⅣhepatitis E virus recombinant open reading frame 3 fusion protein. Determine the cellular localization of hepatitis E virus open reading frame 3 fusion protein by laser scanning confocal microscope. To detect the affect of proliferation of HepG2 cell line with or without actinomycin D caused by hepatitis E virus recombinant open reading frame 3. To detect the change of NF-κB of HepG2 cell line transfected with hepatitis E virus recombinant open reading frame 3 by western blotting.【Results】1. The sera of 78 patients (37.14%) were positive for HEV RNA. They were all shown to be infected with genotype IV HEV by the bi- directional sequencing and phylogenetic analysis. The total mortality is 10%. Among the 210 of sporadic HE patients,179 cases were male (85.2%) and 31 cases were female (14.8%); the ratio of male to female was approximately 5.8:1. In 2007 and 2008, the cases of HE in the first quarter (January-March) accounted for 45.9%(56/122) and 56.8%(50/88) of the whole year. TBil, BUN, and INR are the major risk factors (adjusted OR=1.0009,1.178 and 9.216, respectively) for death in patients with HE. The predictive values of both HEPOD in the first week of sickness were poor, but it improved in the second week and was better able to predict the prognosis of hepatitis E (AUC=0.957).2. We constructed the anti-HEV IgA indirect ELISA assay to evaluate the significance of anti-HEV IgA. The specificity of anti-HEV IgA was 99.6%. Among 245 AHE patients, 84 samples from 84 patients were positive for HEV RNA. The positive rate of anti-HEV IgA, anti-HEV IgM and anti-HEV IgG in 84 samples positive for HEV RNA was 96.3%,97.6% and 88.1% respectively and no sample was negative for anti-HEV IgA and anti-HEV IgM simultaneously. Among 245 AHE patients, we found 9 samples collected from 9 patients in acute period were negative for anti-HEV IgM but positive for anti-HEV IgA and 2 samples were positive for HEV RNA.3. We construct eukaryotic expression vectors(pEGFP-N1 and pcDNA3.1-HisC) for expressing genotype I hepatitis E virus and genotype IV hepatitis E virus recombinant open reading frame 3 fusion protein. The hepatitis E virus open reading frame 3 fusion protein is only localizes in cytoplasm. There is no affect to proliferation of HepG2 cell line without actinomycin D caused by hepatitis E virus recombinant open reading frame 3 and can improve the proliferation of HepG2 cell line with actinomycin D. P65 expressed in cellular nucleus is more in HepG2 cell transfected with hepatitis E virus recombinant open reading frame 3 than HepG2 cell transfecteed with control plasmid.【Conclusion】1. The hepatitis E patients in Hubei province were all shown to be infected with genotype IV HEV. The first quarter (January-March) is the epidemic season for hepatitis E. TBil, BUN, and INR are the major risk factors for death in patients with HE. The predictive value of HEPOD in the second week was good.2. The anti-HEV IgA indirect ELISA assay was constructed. The specificity and sensitivity of anti-HEV IgA was good. Both specificity and sensitivity of combined detection of anti-HEV IgA and anti-HEV IgM was 100% that can improve the accuracy of diagnosis of hepatitis E.3. The hepatitis E virus open reading frame 3 fusion protein is only localizes in cytoplasm. Hepatitis E virus open reading frame 3 and can improve the proliferation of HepG2 cell line treated with actinomycin D maybe through activation of NFκB.更多还原